The binding of a ligand to a protein rarely occurs with the simplicity of a block sliding into an appropriately-shaped hole. Protein and ligand often engage in complementary conformational changes to adapt their shapes to each other. As a result, the structure of a protein bound to its target may differ substantially from the structure of the free protein. Unfortunately, it is virtually impossible to view the binding process in fine structural detail; as a result, most of our knowledge comes from the relatively stable bound and free states. Improving biophysical techniques, however, have brought a change in the way we view some binding events.

Most alterations of conformation during a binding event have historically been interpreted using the induced fit model. In this view, the protein stably maintains the free or “open” structure until it comes into contact with a ligand molecule. This encounter stimulates a conformational change so that the protein adopts the “closed” conformation that tightly holds onto the ligand. Thus, the ligand induces the conformational change necessary to form the bound, closed (BC) structure from the unbound, open (UO) structure, and the intermediate on this path is some kind of bound, open (BO) structure. This model is physically reasonable and has been very successful in interpreting many systems.

However, for the past few decades an increasing amount of evidence has suggested that this is not the whole story. NMR investigations indicated that instead of remaining in a single, well-defined backbone conformation most of the time, many proteins experienced significant changes in their structure while floating free in solution. These results suggested an alternative mechanism of population shift. In this view, the protein actually samples the “closed” conformation (or something very similar) while unbound, and it is this conformation that binds to the ligand. We still go from UO to BC, but now the intermediate is an unbound, closed (UC) structure.

This sounds very arcane, but it is not without functional relevance. Consider, for instance, a protein that is activated by a particular ligand. If we wish to make a drug that binds exclusively to the BC form, then we may experience unforeseen side-effects if our target protein occasionally samples a UC state. It would be useful to have a general idea of what kinds of circumstances are likely to favor a population shift model vs. an induced fit model. That is precisely what Kei-Ichi Okazaki and Shoji Takada aim to provide in an upcoming paper in Proceedings of the National Academy of Sciences(1).

Okazaki and Takada performed a coarse-grained molecular dynamics simulation of glutamine binding protein. In the bound and unbound states they employed a double-well Gō model, a simplified representation of molecular forces, to represent “opening” and “closing”. To switch between these states (i.e. to represent binding) they used a Monte Carlo algorithm. This approach has the advantage of being quick and relatively inexpensive from a computational standpoint, but the results must be interpreted cautiously because the physics of the model are greatly simplified. They observe UO ↔ UC and UC ↔ BC events in this system, but they also observe UO ↔ BO and BO ↔ BC events. This suggests that the simulation will be able to make predictions about both population-shift and induced-fit mechanisms.

In order to try to make some predictions about the circumstances in which a particular mechanism is favored, Okazaki and Takada varied the strength and range of the binding interaction. By monitoring whether the simulated system entered the BC state from BO or UC, they could tell whether the system obeyed the induced-fit or population-shift mechanisms, respectively. They find that as either the strength or the range increase, the induced-fit mechanism is increasingly favored (Figure 4). These results make sense. If the protein regularly samples the closed state while unbound, then the amount of energy needed to reach that state is probably small, so it makes sense to see a population-shift mechanism associated with low-energy binding. Similarly, if a ligand is to associate productively with a non-optimal protein conformation, it makes sense that key interactions will be effective at long range.

From these results Okazaki and Takada suggest that the binding of small hydrophobic ligands is generally likely to proceed via population shift, while the binding of large, charged ligands (such as DNA) will likely proceed via induced fit. They acknowledge, however, that the simulation is limited, particularly in its view of conformational change. Unitary transitions in which the whole protein changes its structure simultaneously are probably not the norm, particularly in the case of very large conformational changes. These changes may instead be stepwise or hierarchical. For instance, a protein or complex recognizing multiple features of a DNA strand may proceed by an apparently induced-fit mechanism, even though each individual binding event more closely resembles population-shift behavior.

An additional limitation of this study is that it considers only one protein, but mechanisms of binding and conformational change may be idiosyncratic properties of particular folds. One could consider the behavior of lymphotactin, which displays clear hallmarks of the population-shift mechanism despite binding to macromolecules (heparin and a GPCR) much larger than itself, as a counterpoint to the predictions developed here. Similarly, the population shift of NtrC involves a charged phosphate group likely to have long-range interactions, although this is a post-translational modification and not a strict ligand-binding event. While the authors point to some examples that match their expectations, overall the data are not unanimously in support of their predictions. Still, the general rules laid out here provide a starting point for experimental work.

Despite the limitations of the simulation, it provides a relatively efficient tool for assessing these processes in other proteins. While no simulation can yet replace experimental data, coarse-grained models like this can serve as a means to formulate testable hypotheses about the energetics of protein-ligand systems.